It is well-known that specific cell lines can be grown in vitro in optimally formulated culture or nutrient media. Examples of some of the culture media developed for special purposes are: RPMI Media 1640 medium for growth of human B-lymphoid cells and malignant cells, Changs medium for growth of amniotic fluid cells, Medium 199 for growth of mouse fibroblast cells, Minimal Essential Medium (MEM) medium, a "minimal" medium for growth of attached mammalian cells, and Leibovitz medium for growth in absence of CO.sub.2. Such various media are distinguished from one another in that they contain critically different components in precise amino acids, vitamins, organic salts, trace elements, and other organic compounds which promote the maximum growth of the cultured cells.
For the growth of mammalian cells chemically defined media are usually supplemented with various sera, preferably fetal calf or newborn calf serum, and other incompletely defined growth factors. A major drawback to serum, however, is that its constituents may vary widely, thereby introducing undefined biological components into the nutrient medium which contributes to the variability of biochemical and cellular events. Additionally, serum is expensive and may result in critical immune reactions in patients if the cells are used for clinical purposes.
The present invention is primarily directed to culturing mammalian hepatocyte cells using chemically defined media that allow long-term expansion of the cell population. The term "chemically defined media" is used in tissue culture to refer to culture media of known chemical composition, both quantitatively and qualitatively, as contrasted with media which contain natural products such as animal serum.
Liver regeneration is achieved primarily by cell division of mature adult hepatocytes as reported by Grisham, J. W., et al., Cancer Res. 22:842 (1962), the disclosure of which is incorporated herein by reference. These cells, or a fraction thereof, have a high capacity for clonal growth, as shown by hepatocyte transplantation experiments in ectopic sites (Jirtle, R. L., et al., Cancer Res. 42:3000 (1982), the disclosure of which is incorporated herein by reference) and in transgenic mouse models (Rhim, J. A., et al., Science 263:1149 (1994), the disclosure of which is incorporated herein by reference). It has been shown in several studies, however, that when liver is stimulated to regenerate while proliferation of mature hepatocytes is suppressed, faculative stem cells emerge and proliferate. See, for example, Thorgeirsson, S. S., et al., Proc. Soc. Exp. Biol. Med. 204:253 (1993), the disclosure of which is incorporated herein by reference. Such cells, sometimes referred to as "oval cells," can mature into hepatocytes in defined animal models or ductular structures composed of cells ("ductular hepatocytes") with mixed hepatocyte and bile duct epithelial markers. See, Gerber, M. A., et al., Amer. J. Path. 110:70 (1983) and Vandersteenhoven, A. M., et al., Arch. Pathol. Lab. Med. 114:403 (1991), the disclosures of which are incorporated herein by reference. Little is known, however, about their origin and about the controls that regulate their phenotypic transitions to hepatocytes or ductular cells.
Despite the high capacity of hepatocytes to proliferate in vivo, directly or via faculative stem cell growth, the conditions that determine their growth potential and their phenotypic transitions are not thoroughly understood because of only limited success in hepatocyte growth in primary culture. It is typically the case that hepatocytes in primary culture under the influence of primary mitogens enter into one or two divisions and then the cells degenerate and die. Heretofore various investigators have failed to develop a medium that allows hepatocytes to both proliferate and survive.
For example, Berry, N. M., et al., J. Cell. Biol. 43:506 (1969), the disclosure of which is incorporated herein by reference, taught the collagenase perfusion technique which allows liver tissue to dissociate into its component cellular elements, based on size. Later, Bissell, D. M., et al., J. Cell. Biol. 59(3):722 (1973) and Bonney, R. J., et al., In Vitro 9:399 (1974), the disclosures of which are incorporated herein by reference, described the first methods for culturing isolated hepatocytes which perhaps survived for one or two days. Long term culture of hepatocytes on collagen gels for a maximum of 7 to 10 days was reported by Michalopoulos, G., et al., Exp. Cell. Res. 94(1):70 (1975), the disclosure of which is incorporated herein by reference. The common characteristic of all of the above-referenced systems is that the hepatocytes in those systems were maintained in culture without there being any evidence of cell proliferation. These systems were, instead, only maintenance cultures of non-proliferating cells for a brief period.
The first successful attempt to initiate DNA synthesis in hepatocytes used the then newly-discovered epidermal growth factor (EGF) as reported by Richman, R. A., et al., Proc. Nat. Acad. Sci. USA 73:3589 (1976), the disclosure of which is incorporated herein by reference. Over the ensuing years several other groups of researchers used EGF as a mitogen for hepatocytes and reported on the mitogenic effects of EGF and their modulation by other factors such as, for example, matrices such as collagen Type I, zinc, and proline.
Hepatocyte growth factor, also known as scatter factor (hereinafter referred to as "HGF" or "HGF/SF") was discovered, cloned and ultimately sequenced by the late 1980's. See Michalopoulos, G., et al., Federation Proceedings 42:1023 (1983); Michalopoulos, G., et al., AACR Proceedings 24:58 (1983); Michalopoulos, G., et al., Cancer Res. 44(10):4414 (1984); and Miyazawa, K., et al., Biochem. Biophys. Res. Comun. 163:967 (1989), the disclosures of which are incorporated herein by reference. HGF/SF was found to be a mitogen for many hepatocytes as well as for epithelial cells. HGF/SF's importance for the liver is due to the fact that it is the trigger for liver regeneration through an endocrine mechanism.
Recently, several studies have shown that HGF/SF, epidermal growth factor ("EGF"), and transforming growth factor .alpha. ("TGF.alpha.") are the primary mitogens for hepatocytes in culture by stimulating limited hepatocyte DNA synthesis in chemically defined media. See, for example, Michalopoulos, G. K., Fed. Am. Soc. Exp. Biochem J. 4:176 (1990), the disclosure of which is incorporated herein by reference. These growth factors were later found to additionally play a role in vivo in liver regeneration after partial hepatectomy. Injection of HGF/SF, TGF.alpha., or EGF in rats induces DNA synthesis in hepatocytes. See, for example, Liu, M. L., et al., Hepatology 19:1521 (1994), the disclosure of which is incorporated herein by reference.
In all of these systems, however, it was reported that hepatocytes entered into DNA synthesis and mitosis for only a limited time, typically 1-3 days. After 1-2 rounds of DNA synthesis and cell division, the cultures degenerated and all the cells were dead in about 7-10 days. Until now, there has also been no documented expansion of the number of hepatocytes in cell culture by adding either EGF or HGF alone, or in combination. The cell replication in cultures containing these growth factors is instead self-limited and the number of hepatocytes that die exceeds the number of hepatocytes that are newly generated. The cell replication in cultures containing other hepatocyte mitogens such as transforming growth factors, such as TGF.alpha., and acidic fibroblast growth factor is similarly self-limited.
More recently, Mitaka, T., et al., Hepatology 13(1):21 (1991); Mitaka, T., et al., Hematology 16(2):440 (1992); Mitaka, T., et al., Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 62:329 (1992); and Mitaka, T., et al., Cancer Res. 53:3145 (1993), the disclosures of which have been incorporated herein by reference, have reported that adding nicotinamide, dexamethasone and EGF to a conventional culture medium resulted in the formation of colonies of small hepatocytes arising in a dense culture of standard size parenchymal hepatocytes. In a further study, Mitaka, T., et al., J. Cell. Physiol. 157:461 (1993), the disclosure of which is incorporated herein by reference, have reported that the numbers of colonies induced by the combinations of EGF+HGF, EGF+TGF-.alpha., and HGF+TGF-.alpha. were not different from those of colonies induced by each mitogen alone. In these studies, however, there was no significant expansion of the total cell population, no evidence of clonal growth, and there was loss of differentiation.
Until now there has been no chemically defined medium, supplemented or not, which is able to support long term proliferation, differentiation, and viability of hepatocytes. While for many purposes the use of an undefined supplement is satisfactory, in cases where studies are made of growth, metabolism, and/or differentiation of cells in culture, it is most desirable to have a supplement that is defined. The introduction of undefined components to a cell culture can contribute to variability, unpredictability, and contamination in study results and applications of cell cultures. The use of defined media is particularly important and advantageous in areas of drug metabolism, artificial organ development, cell transplantation, gene therapy, and basic investigational cell studies.
The above-described limited capacity of hepatocytes to proliferate in primary culture has hindered long-term studies or uses that required long-term viability or proliferation. Applications of hepatocyte cultures to cellular transplantation and gene therapy have thus been hindered. Consequently, there remains a need for a chemically defined medium that will allow hepatocytes to proliferate and survive long term. Among the potential applications for such a medium and the hepatocytes and other cells so cultured are gene therapy, bioartificial organs, cell transplants, drug production, and drug and chemical testing.
As stated above, the current state of the art does not provide a hepatocyte culture system in which hepatocytes expand as a cell population by sustained proliferation, and there is a need for such a system. The present invention provides a fully defined culture medium which allows sustained proliferation and long-term expansion of hepatocytes.